A skeletal muscle ryanodine receptor interaction domain in triadin

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49±0.11-fold and 1.8±0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction.     

Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor

Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca(2)+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH(2)-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca(2)+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca(2)+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 microM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca(2)+ release was smaller and activated at more negative potentials for myotubes expressing the NH(2)-terminal CCD mutations. The shift in the voltage dependence of SR Ca(2)+ release correlated strongly with changes in resting Ca(2)+, SR Ca(2)+ store depletion, and peak voltage-gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca(2)+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca(2)+ leak. These results demonstrate that the NH(2)-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca(2)+ leak, store depletion, and a reduction in voltage-gated Ca(2)+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.     

Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin

In mammalian striated muscles, ryanodine receptor (RyR), triadin, junctin, and calsequestrin form a quaternary complex in the lumen of sarcoplasmic reticulum. Such intermolecular interactions contribute not only to the passive buffering of sarcoplasmic reticulum luminal Ca2+, but also to the active Ca2+ release process during excitation-contraction coupling. Here we tested the hypothesis that specific charged amino acids within the luminal portion of RyR mediate its direct interaction with triadin. Using in vitro binding assay and site-directed mutagenesis, we found that the second intraluminal loop of the skeletal muscle RyR1 (amino acids 4860-4917), but not the first intraluminal loop of RyR1 (amino acids 4581-4640) could bind triadin. Specifically, three negatively charged residues Asp4878, Asp4907, and Glu4908 appear to be critical for the association with triadin. Using deletional approaches, we showed that a KEKE motif of triadin (amino acids 200-232) is essential for the binding to RyR1. Because the second intraluminal loop of RyR has been previously shown to contain the ion-conducting pore as well as the selectivity filter of the Ca2+ release channel, and Asp4878, Asp4907, and Glu4908 residues are predicted to locate at the periphery of the pore assembly of the channel, our data suggest that a physical interaction between RyR1 and triadin could play an active role in the overall Ca2+ release process of excitation-contraction coupling in muscle cells.     

The interaction of spermine with the ryanodine receptor from skeletal muscle.

The effect of polyamines on ryanodine binding activity of junctional sarcoplasmic reticulum membranes is described. Spermine stimulated the binding of ryanodine to its receptor up to 5-fold, with half-maximal stimulation obtained with 3.5 mM. Spermidine and putrescine also stimulated ryanodine binding, but they were about 12-fold less potent. The degree of stimulation is dependent on the NaCl concentration present in the assay medium. Spermine has no effect on the Ca(2+)-dependency of ryanodine binding but it increases the ryanodine binding affinity (Kd) by about 5.6-fold. Both the rate of ryanodine association with, and dissociation from, its binding site were affected by spermine. Spermine also stimulates the photoaffinity labelling by 3-O-(4-benzoyl)benzoyl[alpha-32P]ATP ([alpha-32P]BzATP) of the ryanodine receptor, increasing the BzATP binding affinity. We suggest that the stimulatory effect of spermine on ryanodine binding is due to its specific interaction with the ryanodine receptor. This spermine interaction enabled us to develop a new, one-step, fast and with high yield method for the purification of ryanodine receptor (Shoshan-Barmatz, V. and Zarka, A. (1992) Biochem. J. 284, in press).     

Charged surface area of maurocalcine determines its interaction with the skeletal ryanodine receptor

The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala⁸]MCa, [Ala¹⁹]MCa, [Ala²⁰]MCa, [Ala²²]MCa, [Ala²³]MCa, and [Ala²⁴]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical ²⁴Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys²⁰, Lys²², Arg²³, Arg²⁴, and Lys⁸. Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.     

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.     

Junctin and triadin each activate skeletal ryanodine receptors but junctin alone mediates functional interactions with calsequestrin

Normal Ca²⁺ signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca²⁺ binding protein calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca²⁺ stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level. We show here that purified skeletal ryanodine receptors are similarly activated by purified triadin or purified junctin added to their luminal side, although a lack of competition indicated that the proteins act at independent sites. Surprisingly, triadin and junctin differed markedly in their ability to transmit information between skeletal calsequestrin and ryanodine receptors. Purified calsequestrin inhibited junctin/triadin-associated, or junctin-associated, ryanodine receptors and the calsequestrin re-associated channel complexes were further inhibited when luminal Ca²⁺ fell from 1 mM to ≤100 μM, as seen with native channels (containing endogenous calsequestrin/triadin/junctin). In contrast, skeletal calsequestrin had no effect on the triadin/ryanodine receptor complex and the channel activity of this complex increased when luminal Ca²⁺ fell, as seen with purified channels prior to triadin/calsequestrin re-association. Therefore in this cell free system, junctin alone mediates signals between luminal Ca²⁺, skeletal calsequestrin and skeletal ryanodine receptors and may curtail resting Ca²⁺ leak from the sarcoplasmic reticulum. We suggest that triadin serves a different function which may dominate during excitation–contraction coupling.     

Functional regions of the mouse interleukin-10 receptor cytoplasmic domain.

The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.     

The calmodulin binding region of the skeletal ryanodine receptor acts as a self-modulatory domain

A synthetic peptide (CaMBP) matching amino acids 3614-3643 of the skeletal ryanodine receptor (RyR1) binds to both Ca2+-free calmodulin (CaM) and Ca2+-bound CaM with nanomolar affinity [J. Biol. Chem. 276 (2001) 2069]. We report here that CaMBP increases [3H]ryanodine binding to RyR1 in a dose- and Ca2+-dependent manner; it also induces Ca2+ release from SR vesicles, and increases open probability (P(o)) of single RyR channels reconstituted in planar lipid bilayers. Further, CaMBP removes CaM associated with SR vesicles and increases [3H]ryanodine binding to purified RyR1, suggesting that its mechanism of action is two-fold: it removes endogenous inhibitors and also interacts directly with complementary regions in RyR1. Remarkably, the N-terminus of CaMBP activates RyRs while the C-terminus of CaMBP inhibits RyR activity, suggesting the presence of two discrete functional subdomains within this region. A ryr1 mutant lacking this region, RyR1-Delta3614-3643, was constructed and expressed in dyspedic myoblasts (RyR1-knockout). The depolarization-, caffeine- and 4-chloro-m-cresol (4-CmC)-induced Ca2+ transients in these cells were dramatically reduced compared with cells expressing wild type RyR1. Deletion of the 3614-3643 region also resulted in profound changes in unitary conductance and channel gating. We thus propose that the RyR1 3614-3643 region acts not only as the CaM binding site, but also as an important modulatory domain for RyR1 function.     

Central domain of the human cardiac muscle ryanodine receptor does not mediate interaction with FKBP12.6

The immunophilin, FK506-binding protein (FKBP12), is an essential component of the ryanodine receptor channel complex of skeletal muscle (RyR1) and modulates intracellular calcium signaling from the nedoplasmic reticulum. The cardiac muscle RyR isoform (RyR2) specifically associates with a distinct FKBP isoform, FKBP12.6. Previous studies have led to the proposal that the central domain of RyR1 exclusively mediates the interaction with FKBP12. To characterize the topography of the FKBP 12.6 binding site on the human cardiac RyR2, we have applied complementary protein-protein interaction methods using both in vivo yeast two-hybrid analysis and in vitro immunoprecipitation experiments. Our results indicate an absence of interaction of FKBP12/12.6 with fragments containin the central domain of either RyR1, RyR2, or RyR3. Furthermore, no interaction was detected between FKBP12.6 with a series of overlapping fragments encompassing the entire RyR2, either individually or in multiple combination. We also found that a distinct, alternatively spliced variant of FKBP12.6 was unable to interact with RyR. In contrast, we successfully demonstrated a robust association between the cytoplasmic domain of transforming growth factor-β receptor type I and both FKBP12 and FKBP12.6 in parallel positive control experiments, as well as between native RyR2 and FKBP12.6. These results suggest that the specific interaction of FKBP12.6 with RyR2, and generally of FKBPs with any RyR isoform, is not readily reconstituted by peptide fragments corresponding to central RyR domains. Further structural analysis will be necessary to unravel this intricate signaling system and the current model of FKBP-12-RyR interaction via a single, central RyR, epitope may therefore require revision.     

Retrograde regulation of store-operated calcium channels by the ryanodine receptor-associated protein triadin 95 in rat skeletal myotubes

The 95kDa triadin (or T95), the main skeletal muscle triadin isoform, negatively regulates the mechanism of excitation-contraction coupling. T95 is a ryanodine receptor (RyR)-interacting protein but it also possesses a calsequestrin-interacting domain. RyR and calsequestrin are involved in Ca2+ signalling and, for instance, influence the activity of store-dependent Ca2+ channels (SOC). This work was undertaken to determine whether T95 was able to modulate the entry of Ca2+ through SOC. The experiments were carried out on differentiated rat myotubes over-expressing T95 or DsRed (control cells) by means of an adenovirus infection. Intracellular Ca2+ signals were analyzed using the Ca2+ indicator Fluo-4. The sarco-endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ stores. When applied in the presence of a Ca2+-free medium, thapsigargin elicited transient but long-lasting Fluo-4 responses by elevating the cytoplasmic concentration of Ca2+ ([Ca2+]i). The over-expression of T95 reduced the thapsigargin-dependent [Ca2+]i increase, with respect to control myotubes. Addition of extracellular Ca2+after the depletion of this Ca2+ pool was accompanied by a [Ca2+]i increase that was sensitive to the SOC blockers 2-APB, SKF-96365 and La3+. The over-expression of T95 reduced this Ca2+ influx, without changing its pharmacological properties, showing that T95 over-expression did not alter the properties of the SOC. In conclusion, the RyR-interacting molecule T95, recently shown to inhibit the excitation-contraction coupling, has also the ability to interfere with the skeletal muscle Ca2+ signalling by depressing thapsigargin-dependent Ca2+ release and influx.     

Direct interaction between the reductase domain of endothelial nitric oxide synthase and the ryanodine receptor

We have performed the recombinant expression and purification of the reductase domain of endothelial nitric oxide synthase (eNOS) and used it as a bait in search for interacting proteins present in endothelial cells. Using mass spectrometry of the bound proteins run in a PAGE-SDS gel, we were able to identify the ryanodine receptor (RyR) as a novel eNOS-binding partner. This interaction was confirmed through immunoprecipitation of both RyR and eNOS from endothelial cells and cardiac myocytes. Immunofluorescence data indicated that a subpopulation of eNOS associates with RyR in perinuclear regions of the cell, where eNOS might be responsible for the known nitrosylation of RyR.     

Ca2+对骨骼肌钙释放通道的调节

钙释放通道（calcium release channel）又称Ryanodine受体（RyR）,是细胞内质网膜上介导细胞内钙信号转导的离子通道。RyR1在骨骼肌细胞的兴奋-收缩偶联过程中起重要作用,是肌质网快速释放Ca^2＋的通道。许多调节因素,如一些内源性蛋白（FK结合蛋白、钙调素、钙结合蛋白）和一些离子（Ca^2＋、Mg^2＋）,通过不同的作用位点与RyR1结合,调控RyR1的结构与功能。研究表明,Ca^2＋是众多调节RyR1因素中的核心成分和前提条件,其对RyR1的结构与功能有重要的调控作用。     

Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel

L-type Ca(2+) channel (L-channel) activity of the skeletal muscle dihydropyridine receptor is markedly enhanced by the skeletal muscle isoform of the ryanodine receptor (RyR1) (Nakai, J., R.T. Dirksen, H. T. Nguyen, I.N. Pessah, K.G. Beam, and P.D. Allen. 1996. Nature. 380:72-75.). However, the dependence of the biophysical and pharmacological properties of skeletal L-current on RyR1 has yet to be fully elucidated. Thus, we have evaluated the influence of RyR1 on the properties of macroscopic L-currents and intracellular charge movements in cultured skeletal myotubes derived from normal and "RyR1-knockout" (dyspedic) mice. Compared with normal myotubes, dyspedic myotubes exhibited a 40% reduction in the amount of maximal immobilization-resistant charge movement (Q(max), 7.5 +/- 0.8 and 4.5 +/- 0.4 nC/muF for normal and dyspedic myotubes, respectively) and an approximately fivefold reduction in the ratio of maximal L-channel conductance to charge movement (G(max)/Q(max)). Thus, RyR1 enhances both the expression level and Ca(2+) conducting activity of the skeletal L-channel. For both normal and dyspedic myotubes, the sum of two exponentials was required to fit L-current activation and resulted in extraction of the amplitudes (A(fast) and A(slow)) and time constants (tau(slow) and tau(fast)) for each component of the macroscopic current. In spite of a >10-fold in difference current density, L-currents in normal and dyspedic myotubes exhibited similar relative contributions of fast and slow components (at +40 mV; A(fast)/[A(fast) + A(slow)] approximately 0.25). However, both tau(fast) and tau(slow) were significantly (P < 0.02) faster for myotubes lacking the RyR1 protein (tau(fast), 8.5 +/- 1.2 and 4.4 +/- 0.5 ms; tau(slow), 79.5 +/- 10.5 and 34.6 +/- 3.7 ms at +40 mV for normal and dyspedic myotubes, respectively). In both normal and dyspedic myotubes, (-) Bay K 8644 (5 microM) caused a hyperpolarizing shift (approximately 10 mV) in the voltage dependence of channel activation and an 80% increase in peak L-current. However, the increase in peak L-current correlated with moderate increases in both A(slow) and A(fast) in normal myotubes, but a large increase in only A(fast) in dyspedic myotubes. Equimolar substitution of Ba(2+) for extracellular Ca(2+) increased both A(fast) and A(slow) in normal myotubes. The identical substitution in dyspedic myotubes failed to significantly alter the magnitude of either A(fast) or A(slow). These results demonstrate that RyR1 influences essential properties of skeletal L-channels (expression level, activation kinetics, modulation by dihydropyridine agonist, and divalent conductance) and supports the notion that RyR1 acts as an important allosteric modulator of the skeletal L-channel, analogous to that of a Ca(2+) channel accessory subunit.     

Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium

The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca2+ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-micron intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin- and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co- localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin- and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 micron. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca2+ release channels in atrium may be activated in the absence of a close association with the DHPR.     

Interaction of FKBP12.6 with the cardiac ryanodine receptor C-terminal domain

The ryanodine receptor-calcium release channel complex (RyR) plays a pivotal role in excitation-contraction coupling in skeletal and cardiac muscle. RyR channel activity is modulated by interaction with FK506-binding protein (FKBP), and disruption of the RyR-FKBP association has been implicated in cardiomyopathy, cardiac hypertrophy, and heart failure. Evidence for an interaction between RyR and FKBP is well documented, both in skeletal muscle (RyR1-FKBP12) and in cardiac muscle (RyR2-FKBP12.6), however definition of the FKBP-binding site remains elusive. Early reports proposed interaction of a short RyR central domain with FKBP12/12.6, however this site has been questioned, and recently an alternative FKBP12.6 interaction site has been identified within the N-terminal half of RyR2. In this study, we report evidence for the human RyR2 C-terminal domain as a novel FKBP12.6-binding site. Using competition binding assays, we find that short C-terminal RyR2 fragments can displace bound FKBP12.6 from the native RyR2, although they are unable to exclusively support interaction with FKBP12.6. However, expression of a large RyR2 C-terminal construct in mammalian cells encompassing the pore-forming transmembrane domains exhibits rapamycin-sensitive binding specifically to FKBP12.6 but not to FKBP12. We also obtained some evidence for involvement of the RyR2 N-terminal, but not the central domain, in FKBP12.6 interaction. Our studies suggest that a novel interaction site for FKBP12.6 may be present at the RyR2 C terminus, proximal to the channel pore, a sterically appropriate location that would enable this protein to play a central role in the modulation of this critical ion channel.     

Dantrolene stabilizes domain interactions within the ryanodine receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.     

The interaction of ryanoids with individual ryanodine receptor channels

Ryanodine binds with high affinity and specificity to a class of Ca(2+)-release channels known as ryanodine receptors (RyR). The interaction with RyR results in a dramatic alteration in function with open probability (Po) increasing markedly and rates of ion translocation modified. We have investigated the features of ryanodine that govern the interaction of the ligand with RyR and the mechanisms underlying the subsequent alterations in function by monitoring the effects of congeners and derivatives of ryanodine (ryanoids) on individual RyR2 channels. While the interaction of all tested ryanoids results in an increased Po, the amplitude of the modified conductance state depends upon the structure of the ryanoid. We propose that different rates of cation translocation observed in the various RyR-ryanoid complexes represent different conformations of the channel stabilized by specific conformers of the ligand. On the time scale of a single channel experiment ryanodine binds irreversibly to the channel. However, alterations in structure yield some ryanoids with dissociation rate constants orders of magnitude greater than ryanodine. The probability of occurrence of the RyR-ryanoid complex is sensitive to trans-membrane voltage, with the vast majority of the influence of potential arising from a voltage-driven alteration in the affinity of the ryanoid-binding site.     

High-resolution structure of the membrane-embedded skeletal muscle ryanodine receptor

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